Angiogenesis, defined as the growth of new capillary blood vessels, plays a fundamental role in growth and development. In mature humans, the ability to initiate an angiogenic response is present in all tissues, but is held under strict control. A key regulator of angiogenesis is vascular endothelial growth factor (“VEGF”), also called vascular permeability factor (“VPF”).
VEGF is expressed in abnormally high levels in certain tissues from diseases characterized by aberrant angiogenesis, such as cancers, diabetic retinopathy, psoriasis, age-related macular degeneration, rheumatoid arthritis and other inflammatory diseases. Therefore, agents which selectively decrease the VEGF levels in these tissues can be used to treat cancer and other angiogenic diseases.
Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic-helix-loop-helix-PAS transcription factor consisting of HIF-1 alpha and HIF-1 beta subunits. HIF-1 alpha expression and HIF-1 transcriptional activity increase exponentially as cellular oxygen concentration is decreased. Several dozen target genes that are transactivated by HIF-1 have been identified, including those encoding erythropoietin, glucose transporters, glycolytic enzymes, and VEGF. Semenza GL (1999), Ann. Rev. Cell. Dev. Biol. 15: 551-578.
Loss of p53 in tumor cells enhances HIF-1 alpha levels and augments HIF-1-dependent transcriptional activation of VEGF in response to hypoxia. Forced expression of HIF-1 alpha in p53-expressing tumor cells increases hypoxia-induced VEGF expression and augments neovascularization and growth of tumor xenografts. These results indicate that amplification of normal HIF-1-dependent responses to hypoxia via loss of p53 function contributes to the angiogenic switch during tumorigenesis. Ravi R. et al. (2000), Genes Dev. 14: 34-44.
RNA interference (“RNAi”) is a method of post-transcriptional gene regulation that is conserved throughout many eukaryotic organisms. RNAi is induced by short (i.e., <30 nucleotide) double stranded RNA (“dsRNA”) molecules which are present in the cell (Fire A et al. (1998), Nature 391: 806-811). These short dsRNA molecules, called “short interfering RNA” or “siRNA,” cause the destruction of messenger RNAs (“mRNAs”) which share sequence homology with the siRNA to within one nucleotide resolution (Elbashir S M et al. (2001), Genes Dev, 15: 188-200). It is believed that the siRNA and the targeted mRNA bind to an RNA-induced silencing complex (“RISC”), which cleaves the targeted mRNA. The siRNA is apparently recycled much like a multiple-turnover enzyme, with 1 siRNA molecule capable of inducing cleavage of approximately 1000 mRNA molecules. siRNA-mediated RNAi is therefore more effective than other currently available technologies for inhibiting expression of a target gene.
Elbashir S M et al. (2001), supra, has shown that synthetic siRNA of 21 and 22 nucleotides in length, and which have short 3′ overhangs, can induce RNAi of target mRNA in a Drosophila cell lysate. Cultured mammalian cells also exhibit RNAi with synthetic siRNA (Elbashir S M et al. (2001) Nature, 411: 494-498), and RNAi induced by synthetic siRNA has recently been shown in living mice (McCaffrey A P et al. (2002), Nature, 418: 38-39; Xia H et al. (2002), Nat. Biotech. 20: 1006-1010). The therapeutic potential of siRNA-mediated RNAi has been demonstrated by several recent in vitro studies, including the siRNA-directed inhibition of HIV-1 infection (Novina C D et al. (2002), Nat. Med. 8: 681-686) and reduction of neurotoxic polyglutamine disease protein expression (Xia H et al. (2002), supra). Therapeutic RNAi has also been demonstrated in human cancer cells by Alan Gewirtz, as described in published U.S. patent application US 2002/0173478.
It has now been found that siRNA-induced RNAi of HIF-1 alpha results in the destruction of HIF-1 alpha mRNA, with a concomitant reduction in VEGF expression and inhibition of angiogenesis.